sp1 coding sequence  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer

doi: 10.1038/s41419-025-08143-5

Figure Lengend Snippet: MDA-MB-231 cells were transfected with oligonucleotides targeting NR4A1 ( A ), NR4A2 ( B ), Sp1 ( C ), Sp4 ( D ) and Sp3 ( E ), and after 72 h, whole cell lysates were analyzed by Western blots and quantified as outlined in the “Methods”. F Effects of Sp1 overexpression on CD71 were obtained by transfection of the p CVM-Sp1 expression plasmid and subsequent western blot analysis of whole cell lysates and quantified as outlined in the “Methods”. Cells were treated with 10 µM DIM-3,5-CI 2 ( G ) and DIM-3-CI-5-CF 3 ( H ) for up to 24 h, and effects on CD71, Sp1 and Sp4 (separate blot) were determined by Western blots of whole cell lysates and quantified as outlined in the “Methods”.

Article Snippet: The plasmid containing Sp1 coding sequence (Addgene plasmid # 12097) and empty vector as negative control (Addgene plasmid # 20783) using LipofectamineTM 3000 reagent (Invitrogen, # L3000008) according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Over Expression, Expressing, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer

doi: 10.1038/s41419-025-08143-5

Figure Lengend Snippet: Interactions of NR4A1 ( A ), NR4A2 ( B ), Sp1 ( C ) and Sp4 ( D ) with the CD71 gene promoter containing the Sp binding region was determined after treatment with DIM-3,5 compounds in a ChIP assay (in triplicate) as outlined in the “Methods section”. E Summary of the CD71 gene promoter, Sp binding sites (−1576 to −1566) and the primers used for detecting protein interactions in this region of the promoter, along with the overall mechanism of ferroptosis induction. Results are expressed as means ± SD for replicate (3) determinations for each treatment group ( A – D ) and significant ( p < 0.05) induction is indicated (*).

Article Snippet: The plasmid containing Sp1 coding sequence (Addgene plasmid # 12097) and empty vector as negative control (Addgene plasmid # 20783) using LipofectamineTM 3000 reagent (Invitrogen, # L3000008) according to the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Nature

Article Title: Promoter-bound METTL3 maintains myeloid leukaemia by m 6 A-dependent translation control.

doi: 10.1038/nature24678

Figure Lengend Snippet: Figure 3 | Transcripts derived from METTL3-bound promoters harbour m6A within their coding sequence (CDS). a, Genomic visualization of the m6A immunoprecipitation normalized signal in METTL3-KD or control MOLM13 cells on the SP1 transcript (upper tracks), along with the genomic visualization of the METTL3 ChIP–seq. b, Pie charts of the distribution of METTL3-dependent m6A peaks within the whole transcriptome or METTL3 chromatin target mRNAs. c, m6A immunoprecipitation followed by qPCR for m6A peaks of SP1 and SP2. The plot shows the m6A immunoprecipitation signal over total input in MOLM13 cells expressing a control shRNA or shRNAs targeting CEBPZ. Mean + s.d. of three technical replicates shown; experiment

Article Snippet: Rescue experiments. cDNA was obtained by reverse transcription of MOLM13 cell RNA with Supercript III (ThermoFisher Scientific), then the SP1 full-length coding sequence was amplified by PCR and cloned into pHIV-ZsGreen plasmid (Addgene 18121) by Gibson assembly (Gibson Assembly Cloning Kit, NEB), using the HpaI site.

Techniques: Derivative Assay, Sequencing, Immunoprecipitation, Control, ChIP-sequencing, Expressing, shRNA